Transgenic animal for production of antibodies having minimal CDRS

ABSTRACT

A transgenic animal is provided. In certain embodiments, the transgenic animal comprises a genome comprising: an immunoglobulin light chain locus comprising: a) a functional immunoglobulin light chain gene comprising a transcribed variable region encoding: i. light chain CDR1, CDR2 and CDR3 regions that are composed of 2 to 5 different amino acids; and ii. a light chain framework; and, operably linked to the functional immunoglobulin light chain gene: b) a plurality of pseudogene light chain variable regions each encoding: i. light chain CDR1, CDR2 and CDR3 regions that are composed of the same 2 to 5 different amino acids as the CDRs of the functional gene; and ii. a light chain framework that is identical in amino acid sequence to the light chain framework of the transcribed variable region.

CROSS-REFERENCING

This application is a continuation of U.S. patent application Ser. No.15/188,724, filed on Jun. 21, 2016, now issued as U.S. Pat. No.9,549,538, which is a divisional of U.S. patent application Ser. No.14/057,820, filed on Oct. 18, 2013, now issued as U.S. Pat. No.9,404,125, which is a divisional of U.S. patent application Ser. No.12/854,722, filed on Aug. 11, 2010, now issued as U.S. Pat. No.8,592,644, which claims the priority benefit of U.S. provisionalapplication Ser. No. 61/274,319, filed Aug. 13, 2009, all of which areincorporated by reference in its herein in their entirety.

BACKGROUND

Antibodies are proteins that bind a specific antigen. Generally,antibodies are specific for their targets, have the ability to mediateimmune effector mechanisms, and have a long half-life in serum. Suchproperties make antibodies powerful therapeutics. Monoclonal antibodiesare used therapeutically for the treatment of a variety of conditionsincluding cancer, inflammation, and cardiovascular disease. There arecurrently over twenty therapeutic antibody products on the market andhundreds in development.

There is a constant need for new antibodies and methods for making thesame.

SUMMARY

A transgenic non-human animal is provided. In certain embodiments, thetransgenic animal comprises a genome comprising: an immunoglobulin lightchain locus comprising: a) a functional immunoglobulin light chain genecomprising a transcribed variable region encoding: i. light chain CDR1,CDR2 and CDR3 regions that are composed of 2 to 5 different amino acids;and ii. a light chain framework; and, operably linked to the functionalimmunoglobulin light chain gene: b) a plurality of pseudogene lightchain variable regions each encoding: i. light chain CDR1, CDR2 and CDR3regions that are composed of the same 2 to 5 different amino acids asthe CDRs of the functional gene; and ii. a light chain framework that isidentical in amino acid sequence to the light chain framework of thetranscribed variable region, where the plurality of pseudogene lightchain variable regions donate nucleotide sequence to the transcribedvariable region of the functional immunoglobulin light chain gene bygene conversion in the transgenic animal.

In addition or as an alternative to the above, the transgenic animal maycomprise an immunoglobulin heavy chain locus comprising: a) a functionalimmunoglobulin heavy chain gene comprising a transcribed variable regionencoding: i. heavy chain CDR1, CDR2 and CDR3 regions that are composedof 2 to 5 different amino acids (e.g., the same 2 to 5 amino acids asthe light chain); and ii. a heavy chain framework; and, operably linkedto the functional immunoglobulin heavy chain gene: b) a plurality ofpseudogene heavy chain variable regions each encoding: i. heavy chainCDR1, CDR2 and CDR3 regions that are composed of the same 2 to 5different amino acids as the functional gene; and ii. a heavy chainframework that is identical in amino acid sequence to the heavy chainframework of the transcribed variable region, where the plurality ofpseudogene heavy chain variable regions donate nucleotide sequence tothe transcribed variable region of the functional immunoglobulin heavychain gene by gene conversion in the transgenic animal.

Also provided are methods of producing and method of using thetransgenic animal, as well as antibody compositions produced by thesame.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 schematically illustrates a strategy for deleting a chickenimmunoglobulin light chain locus.

FIG. 2 schematically illustrates a strategy for adding a synthetic arrayof variable region-encoding pseudogenes to a chicken immunoglobulinlight chain locus after deletion of the endogenous chickenimmunoglobulin light chain gene.

FIG. 3 schematically illustrates a strategy for constructing an array ofvariable region-encoding pseudogenes.

FIG. 4 schematically illustrates a strategy for constructing a vectorfor inserting an array of variable region-encoding pseudogenes.

FIG. 5 schematically illustrates a strategy to place an attP site in thechicken IgL locus.

FIG. 6 show the results of PCR analysis of chicken IgL knockout andknock-in clones.

FIG. 7 schematically illustrates a strategy for making knock-ins.

FIGS. 8A and 8B illustrates examples of gene conversion events for CDR1.SEQ ID NOS: 1-6.

FIG. 9 is a table showing the expression levels of various heavy andlight chain sequences.

FIG. 10 are graphs showing the stability of various antibodies after anextended incubation period.

FIG. 11 shows the nucleotide sequence and encoded amino acid sequence ofthe E6 (light chain). SEQ ID NOS: 53 and 54.

FIG. 12 shows the nucleotide sequence and encoded amino acid sequence ofthe C3 (heavy chain). SEQ ID NOS: 55 and 56.

DEFINITIONS

The terms “determining”, “measuring”, “evaluating”, “assessing” and“assaying” are used interchangeably herein to refer to any form ofmeasurement, and include determining if an element is present or not.These terms include both quantitative and/or qualitative determinations.Assessing may be relative or absolute. “Determining the presence of”includes determining the amount of something present, as well asdetermining whether it is present or absent.

The term “gene” refers to a nucleic acid sequence comprised of apromoter region, a coding sequence, and a 3′UTR.

The terms “protein” and “polypeptide” are used interchangeably herein.

A “leader sequence” is a sequence of amino acids present at theN-terminal portion of a protein which facilitates the secretion of themature form of the protein from the cell. The definition of a signalsequence is a functional one. The mature form of the extracellularprotein lacks the signal sequence, which is cleaved off during thesecretion process.

The term “nucleic acid” encompasses DNA, RNA, single stranded or doublestranded and chemical modifications thereof. The terms “nucleic acid”and “polynucleotide” are used interchangeably herein.

A “non-human” animal refers to any animal of a species that is nothuman.

The term “progeny” or “off-spring” refers to any and all futuregenerations derived and descending from a particular animal. Thus,progeny of any successive generation are included herein such that theprogeny, the F1, F2, F3, generations and so on are included in thisdefinition.

The phrase “transgenic animal” refers to an animal comprising cellscontaining foreign nucleic acid (i.e., recombinant nucleic acid that isnot native to the animal). The foreign nucleic acid may be present inall cells of the animal or in some but not all cells of the animal. Theforeign nucleic acid molecule is called a “transgene” and may containone or many genes, cDNA, etc. By inserting a transgene into a fertilizedoocyte or cells from the early embryo, the resulting transgenic animalmay be fully transgenic and able to transmit the foreign nucleic acidstably in its germline. Alternatively, a foreign nucleic acid may beintroduced by transferring, e.g., implanting, a recombinant cell ortissue containing the same into an animal to produce a partiallytransgenic animal. Alternatively, a transgenic animal may be produced bytransfer of a nucleus from a genetically modified somatic cell or bytransfer of a genetically modified pluripotential cell such as anembryonic stem cell or a primordial germ cell.

The term “intron” refers to a sequence of DNA found in the middle ofmany gene sequences in most eukaryotes. These intron sequences aretranscribed, but removed from within the pre-mRNA transcript before themRNA is translated into a protein. This process of intron removal occursby splicing together of the sequences (exons) on either side of theintron.

The term “operably-linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably-linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Similarly, when an intron isoperably-linked to a coding sequence, the intron is spliced out of themRNA to provide for expression of the coding sequence. In the context ofgene conversion, two nucleic acids sequences are operably linked if onesequence can “donate” sequence to the other by gene conversion. If twosequences are unlinked in that one can donate sequence to the other viagene conversion, the donating sequences may be upstream or downstream ofthe other, and the two sequences may be proximal to each other, i.e., inthat there are no other intervening genes. “Unlinked” means that theassociated genetic elements are not closely associated with one anotherand the function of one does not affect the other.

The terms “upstream” and “downstream” are used with reference to thedirection of transcription.

The term “pseudogene” is used to describe an untranscribed nucleic acidregion that contains an open reading frame that may or may not contain astart and/or a stop codon. An amino acid sequence may be “encoded” by apseudogene in the sense that the nucleotide sequence of the open readingframe can be translated in silico to produce an amino acid sequence. Inthe context of the heavy and light chain immunoglobulin loci,pseudogenes do not contain promoter regions, recombination signalsequences or leader sequences.

The term “homozygous” indicates that identical alleles reside at thesame loci on homologous chromosomes. In contrast, “heterozygous”indicates that different alleles reside at the same loci on homologouschromosomes. A transgenic animal may be homozygous or heterozygous for atransgene.

The term “endogenous”, with reference to a gene, indicates that the geneis native to a cell, i.e., the gene is present at a particular locus inthe genome of a non-modified cell. An endogenous gene may be a wild typegene present at that locus in a wild type cell (as found in nature). Anendogenous gene may be a modified endogenous gene if it is present atthe same locus in the genome as a wild type gene. An example of such amodified endogenous gene is a gene into which a foreign nucleic acid isinserted. An endogenous gene may be present in the nuclear genome,mitochondrial genome etc.

The term “construct” refers to a recombinant nucleic acid, generallyrecombinant DNA, that has been generated for the purpose of theexpression of a specific nucleotide sequence(s), or is to be used in theconstruction of other recombinant nucleotide sequences. A constructmight be present in a vector or in a genome.

The term “recombinant” refers to a polynucleotide or polypeptide thatdoes not naturally occur in a host cell. A recombinant molecule maycontain two or more naturally-occurring sequences that are linkedtogether in a way that does not occur naturally. A recombinant cellcontains a recombinant polynucleotide or polypeptide. If a cell receivesa recombinant nucleic acid, the nucleic acid is “exogenous” to the cell.

The term “selectable marker” refers to a protein capable of expressionin a host that allows for ease of selection of those hosts containing anintroduced nucleic acid or vector. Examples of selectable markersinclude, but are not limited to, proteins that confer resistance toantimicrobial agents (e.g., hygromycin, bleomycin, or chloramphenicol),proteins that confer a metabolic advantage, such as a nutritionaladvantage on the host cell, as well as proteins that confer a functionalor phenotypic advantage (e.g., cell division) on a cell.

The term “expression”, as used herein, refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or ‘transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell wherein the nucleicacid sequence may be incorporated into the genome of the cell (e.g.,chromosome, plasmid, plastid, or mitochondrial DNA), converted into anautonomous replicon, or transiently expressed (e.g., transfected mRNA).

The term “replacing”, in the context of replacing one genetic locus withanother, refers to a single step protocol or multiple step protocol.

The term “coding sequence” refers to a nucleic acid sequence that oncetranscribed and translated produces a protein, for example, in vivo,when placed under the control of appropriate regulatory elements. Acoding sequence as used herein may have a continuous ORF or might havean ORF interrupted by the presence of introns or non-coding sequences.In this embodiment, the non-coding sequences are spliced out from thepre-mRNA to produce a mature mRNA. Pseudogenes may contain anuntranscribed coding sequence.

The term “in reverse orientation to” refers to coding sequences that areon different strands. For example, if a transcribed region is describedas being in reverse orientation to a pseudogene, then the amino acidsequence encoded by the transcribed region is encoded by the top orbottom strand and the amino acid sequence encoded by the pseudogene isencoded by the other strand relative to the transcribed region. Asillustrated in FIG. 8, the orientation of a coding sequence may beindicated by an arrow.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. These terms are well understood by those in the field, and referto a protein consisting of one or more polypeptides that specificallybinds an antigen. One form of antibody constitutes the basic structuralunit of an antibody. This form is a tetramer and consists of twoidentical pairs of antibody chains, each pair having one light and oneheavy chain. In each pair, the light and heavy chain variable regionsare together responsible for binding to an antigen, and the constantregions are responsible for the antibody effector functions.

The recognized immunoglobulin polypeptides include the kappa and lambdalight chains and the alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta,epsilon and mu heavy chains or equivalents in other species. Full-lengthimmunoglobulin “light chains” (of about 25 kDa or about 214 amino acids)comprise a variable region of about 110 amino acids at the NH₂-terminusand a kappa or lambda constant region at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (of about 50 kDa or about 446 aminoacids), similarly comprise a variable region (of about 116 amino acids)and one of the aforementioned heavy chain constant regions, e.g., gamma(of about 330 amino acids).

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,single-chain antibodies, and fusion proteins comprising anantigen-binding portion of an antibody and a non-antibody protein. Theantibodies may be detectably labeled, e.g., with a radioisotope, anenzyme which generates a detectable product, a fluorescent protein, andthe like. The antibodies may be further conjugated to other moieties,such as members of specific binding pairs, e.g., biotin (member ofbiotin-avidin specific binding pair), and the like. The antibodies mayalso be bound to a solid support, including, but not limited to,polystyrene plates or beads, and the like. Also encompassed by the termare Fab′, Fv, F(ab′)₂, and or other antibody fragments that retainspecific binding to antigen, and monoclonal antibodies.

Antibodies may exist in a variety of other forms including, for example,Fv, Fab, and (Fab′)₂, as well as bi-functional (i.e. bi-specific) hybridantibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987))and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci.U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426(1988), which are incorporated herein by reference). (See, generally,Hood et al., “Immunology”, Benjamin, N.Y., 2nd ed. (1984), andHunkapiller and Hood, Nature, 323, 15-16 (1986),).

An immunoglobulin light or heavy chain variable region consists of a“framework” region (FR) interrupted by three hypervariable regions, alsocalled “complementarity determining regions” or “CDRs”. The extent ofthe framework region and CDRs have been precisely defined (see, Lefrancet al, IMGT, the international ImMunoGeneTics information system.Nucleic Acids Res. 2009 vol. 37 (Database issue): D1006-12. Epub 2008Oct. 31; see worldwide website of imgt.org and referred to hereinafteras the “IMGT system”)). The numbering of all antibody amino acidsequences discussed herein conforms to the IMGT system. The sequences ofthe framework regions of different light or heavy chains are relativelyconserved within a species. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs. The CDRs are primarilyresponsible for binding to an epitope of an antigen.

Chimeric antibodies are antibodies whose light and heavy chain geneshave been constructed, typically by genetic engineering, from antibodyvariable and constant region genes belonging to different species. Forexample, the variable segments of the genes from a chicken or rabbitmonoclonal antibody may be joined to human constant segments, such asgamma 1 and gamma 3. An example of a therapeutic chimeric antibody is ahybrid protein composed of the variable or antigen-binding domain from achicken or rabbit antibody and the constant or effector domain from ahuman antibody (e.g., the anti-Tac chimeric antibody made by the cellsof A.T.C.C. deposit Accession No. CRL 9688), although other mammalianspecies may be used.

As used herein, the term “human framework” refers to a framework thathas an amino acid sequence that is at least 90% identical, e.g., atleast 95%, at least 98% or at least 99% identical to the amino acidsequence of a human antibody, e.g., the amino acid sequence of a humangerm-line sequence of an antibody. In certain cases, a human frameworkmay be a fully human framework, in which case the framework has an aminoacid sequence that is identical to that of a human antibody, e.g., agerm-line antibody.

As used herein, the term “humanized antibody” or “humanizedimmunoglobulin” refers to a non-human antibody containing one or moreamino acids (in a framework region, a constant region or a CDR, forexample) that have been substituted with a correspondingly positionedamino acid from a human antibody. In general, humanized antibodies areexpected to produce a reduced immune response in a human host, ascompared to a non-humanized version of the same antibody.

It is understood that the humanized antibodies designed and produced bythe present method may have additional conservative amino acidsubstitutions which have substantially no effect on antigen binding orother antibody functions. By conservative substitutions is intendedcombinations such as those from the following groups: gly, ala; val,ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Aminoacids that are not present in the same group are “substantiallydifferent” amino acids.

The term “specific binding” refers to the ability of an antibody topreferentially bind to a particular analyte that is present in ahomogeneous mixture of different analytes. In certain embodiments, aspecific binding interaction will discriminate between desirable andundesirable analytes in a sample, in some embodiments more than about 10to 100-fold or more (e.g., more than about 1000- or 10,000-fold).

In certain embodiments, the affinity between an antibody and analytewhen they are specifically bound in an antibody/analyte complex ischaracterized by a K_(D) (dissociation constant) of less than 10⁻⁶ M,less than 10⁻⁷ M, less than 10⁻⁸ M, less than 10⁻⁹ M, less than 10⁻⁹ M,less than 10⁻¹¹ M, or less than about 10⁻¹² M or less.

A “variable region” of a heavy or light antibody chain is an N-terminalmature domain of the chain that contains CDR1, CDR2 and CD3, andframework regions. The heavy and light chain of an antibody both containa variable domain. All domains, CDRs and residue numbers are assigned onthe basis of sequence alignments and structural knowledge.Identification and numbering of framework and CDR residues is as definedby the IMGT system.

VH is the variable domain of an antibody heavy chain. VL is the variabledomain of an antibody light chain.

As used herein the term “isolated,” when used in the context of anisolated antibody, refers to an antibody of interest that is at least60% free, at least 75% free, at least 90% free, at least 95% free, atleast 98% free, and even at least 99% free from other components withwhich the antibody is associated with prior to purification.

The terms “treatment” “treating” and the like are used herein to referto any treatment of any disease or condition in a mammal, e.g.particularly a human or a mouse, and includes: a) preventing a disease,condition, or symptom of a disease or condition from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; b) inhibiting a disease, condition, or symptomof a disease or condition, e.g., arresting its development and/ordelaying its onset or manifestation in the patient; and/or c) relievinga disease, condition, or symptom of a disease or condition, e.g.,causing regression of the condition or disease and/or its symptoms.

The terms “subject,” “host,” “patient,” and “individual” are usedinterchangeably herein to refer to any mammalian subject for whomdiagnosis or therapy is desired, particularly humans. Other subjects mayinclude cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses,and so on.

A “natural” antibody is an antibody in which the heavy and lightimmunoglobulins of the antibody have been naturally selected by theimmune system of a multi-cellular organism, as opposed to unnaturallypaired antibodies made by e.g. phage display. As such, the certainantibodies do not contain any viral (e.g., bacteriophage M13)-derivedsequences. Spleen, lymph nodes and bone marrow are examples of tissuesthat produce natural antibodies in an animal.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or ‘transformation”, or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell wherein the nucleicacid sequence may be present in the cell transiently or may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid, or mitochondrial DNA), converted into an autonomous replicon.

The term “plurality” refers to at least 2, at least 5, at least 10, atleast 20, at least 50, at least 100, at least 200, at least 500, atleast 1000, at least 2000, at least 5000, or at least 10,000 or at least50,000 or more. In certain cases, a plurality includes at least 10 to50. In other embodiments, a plurality may be at least 50 to 1,000.

Further definitions may be elsewhere in this disclosure.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

A transgenic animal is provided. In certain embodiments, the transgenicanimal comprises a genome comprising: an immunoglobulin locuscomprising: a) a functional immunoglobulin gene comprising a transcribedvariable region encoding: i. CDR1, CDR2 and CDR3 regions that arecomposed of 2 to 5 different amino acids; and ii. a framework region;and, operably linked to the functional immunoglobulin gene: b) aplurality of pseudogene light chain variable regions each encoding: i.CDR1, CDR2 and CDR3 regions that are composed of the same 2 to 5different amino acids as the functional gene; and ii. a framework regionthat is identical in amino acid sequence to the framework region of thetranscribed variable region, where the plurality of pseudogene variableregions donate nucleotide sequence to the transcribed variable region ofthe functional immunoglobulin gene by gene conversion in the transgenicanimal. The immunoglobulin locus may be an immunoglobulin light chainlocus or an immunoglobulin heavy chain locus. In certain cases, theanimal may contain both heavy and light chain loci as described herein.

Before the present subject invention is described further, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, and as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of cells and reference to “a candidate agent”includes reference to one or more candidate agents and equivalentsthereof known to those skilled in the art, and so forth. It is furthernoted that the claims may be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology as “solely”, “only” and the like inconnection with the recitation of claim elements, or use of a “negative”limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Transgenic Animals

As noted above, a transgenic animal is provided. In certain embodiments,the animal may be any non-human animal that employs gene conversion fordeveloping their primary antigen repertoire and, as such, the animal maybe any of a variety of different animals. In one embodiment, the animalmay be a bird, e.g., a member of the order Galliformes such as a chickenor turkey, or a member of the order Anseriformes such as a duck orgoose, or a mammal, e.g., a lagamorph such as rabbit, or a farm animalsuch as a cow, sheep, pig or goat. In particular embodiments, thetransgenic animal may be a non-rodent (e.g., non-mouse or non-rat),non-primate transgenic animal.

Some of this disclosure relates to a transgenic chicken containing oneor more transgenes that encode an array of synthetic variable regions.Since the nucleotide sequences of the immunoglobulin loci of manyanimals are known, as are methods for modifying the genome of suchanimals, the general concepts described below may be readily adapted toany suitable animal, i.e., any animal that employs gene conversion fordeveloping their primary antigen repertoire. The generation of antibodydiversity by gene conversion between the variable region of atranscribed immunoglobulin heavy or light chain gene and operably linked(upstream) pseudo-genes that contain different variable regions isdescribed in a variety of publications such as, for example, Butler(Rev. Sci. Tech. 1998 17: 43-70), Bucchini (Nature 1987 326: 409-11),Knight (Adv. Immunol. 1994 56: 179-218), Langman (Res. Immunol. 1993144: 422-46), Masteller (Int. Rev. Immunol. 1997 15: 185-206), Reynaud(Cell 1989 59: 171-83) and Ratcliffe (Dev. Comp. Immunol. 2006 30:101-118).

In certain embodiments, the transgenic animal contains a functionalimmunoglobulin light chain gene that is expressed (i.e., transcribed toproduce an mRNA that is subsequently translated) to produce a lightchain of an antibody, and, operably linked to (which, in the case ischicken and many other species is immediately upstream of) thefunctional light chain gene, a plurality of different pseudogene lightchain variable regions, where the variable regions of the pseudogenesare operably linked to the functional immunoglobulin light chain in thatthey the alter the sequence of the functional immunoglobulin light chaingene by gene conversion (i.e., by substituting a sequence of thefunctional immunoglobulin light chain gene variable region with asequence of a pseudogene variable region). In the transgenic animal,gene conversion between the functional immunoglobulin light chain genevariable region and a pseudogene variable region alters the sequence ofthe functional immunoglobulin light chain gene variable region by aslittle as a single codon up to the entire length of the variable region.In certain cases a pseudogene variable region may donate the sequence ofat least one CDR (e.g., CDR1, CDR2 or CDR3) from a pseudogene variableregion in to the variable region of the functional gene. The lightchains of the antibodies produced by the transgenic animal are thereforeencoded by whatever sequence is donated from the pseudogene variableregions into the variable region of the functional light chain gene.

Likewise, the transgenic animal may also contain an a functionalimmunoglobulin heavy chain gene that is transcribed and translated toproduce a heavy chain of an antibody, and, operably linked to (e.g.,immediately upstream of) the functional heavy chain gene, a plurality ofdifferent pseudogene heavy chain variable regions, where the variableregions of the pseudogenes are operably linked to the functionalimmunoglobulin light chain in that they alter the sequence of thefunctional immunoglobulin heavy chain gene by gene conversion. In thetransgenic animal, gene conversion between the functional immunoglobulinheavy chain gene variable region and a pseudogene variable region altersthe sequence of the functional immunoglobulin heavy chain gene variableregion by as little as a single codon up to the entire length of thevariable region. In certain cases may a pseudogene variable region maydonate the sequence of at least one CDR (e.g., CDR1, CDR2 or CDR3) froma pseudogene variable region to the variable region of the functionalgene. The heavy chains of the antibodies produced by the transgenicanimal are therefore encoded by whatever sequence is donated from thepseudogene variable regions into the variable region of the functionalheavy chain gene.

The antibodies produced by the transgenic animal are therefore encodedby whatever sequences are donated from the pseudogene variable regionsto the variable region of the functional gene. Since different sequencesare donated in different cells of the animal, the antibody repertoire ofthe animal is determined by which sequences are donated from thepseudogene variable regions to the variable region of the functionalgene.

In particular embodiments, the framework encoded by the variable regionpseudogenes is identical in amino acid sequence to the framework regionof the functional gene to which the pseudogenes are operably linked. Inother words, the amino acid sequence of all of the FR1 regions encodedby the pseudogenes may be identical to the FR1 region encoded by thetranscribed variable domain, the amino acid sequence of all of the FR2regions encoded by the pseudogenes may be identical to the FR2 regionencoded by the transcribed variable domain, the amino acid sequence ofall of the FR3 regions encoded by the pseudogenes may be identical tothe FR3 region encoded by the transcribed variable domain and the aminoacid sequence of all of the FR4 regions encoded by the pseudogenes maybe identical to the FR4 region encoded by the transcribed variabledomain, thereby allowing the production of an antibody with a definedheavy and/or light chain framework.

In particular embodiments, the nucleotide sequences encoding theframework of the variable region pseudogenes may be identical to thenucleotide sequences encoding the framework of the functional gene towhich the pseudogenes are operably linked. In other words, thenucleotide sequence encoding all of the FR1 regions in the pseudogenesmay be identical to the nucleotide sequence encoding the FR1 region ofthe transcribed variable domain, the nucleotide sequence encoding all ofthe FR2 regions in the pseudogenes may be identical to the nucleotidesequence encoding the FR2 region of the transcribed variable domain, thenucleotide sequence encoding all of the FR3 regions in the pseudogenesmay be identical to the nucleotide sequence encoding the FR3 region ofthe transcribed variable domain and the nucleotide sequence encoding allof the FR4 regions in the pseudogenes may be identical to the nucleotidesequence encoding the FR4 region of the transcribed variable domain,thereby resulting in an functional gene with a defined nucleotidesequence.

The chosen framework sequence may be human, e.g., have a sequence thatis at least 90%, at least 95%, at least 98%, at least 99% or 100%identical to the germ-line sequence of a human antibody, therebyallowing production of an antibody containing a human framework.

In particular embodiments, the light chain germline sequence is selectedfrom human VK sequences including, but not limited to, A1, A10, A11,A14, A17, A18, A19, A2, A20, A23, A26, A27, A3, A30, A5, A7, B2, B3, L1,L10, L11, L12, L14, L15, L16, L18, L19, L2, L20, L22, L23, L24, L25,L4/18a, L5, L6, L8, L9, O1, O11, O12, O14, O18, O2, O4, and O8. Incertain embodiments, the light chain human germline framework isselected from V1-11, V1-13, V1-16, V1-17, V1-18, V1-19, V1-2, V1-20,V1-22, V1-3, V1-4, V1-5, V1-7, V1-9, V2-1, V2-11, V2-13, V2-14, V2-15,V2-17, V2-19, V2-6, V2-7, V2-8, V3-2, V3-3, V3-4, V4-1, V4-2, V4-3,V4-4, V4-6, V5-1, V5-2, V5-4, and V5-6. See PCT WO 2005/005604 for adescription of the different germline sequences.

In other embodiments, the heavy chain human germline framework isselected from VH1-18, VH1-2, VH1-24, VH1-3, VH1-45, VH1-46, VH1-58,VH1-69, VH1-8, VH2-26, VH2-5, VH2-70, VH3-11, VH3-13, VH3-15, VH3-16,VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48,VH3-49, VH3-53, VH3-64, VH3-66, VH3-7, VH3-72, VH3-73, VH3-74, VH3-9,VH4-28, VH4-31, VH4-34, VH4-39, VH4-4, VH4-59, VH4-61, VH5-51, VH6-1,and VH7-81. See PCT WO 2005/005604 for a description of the differentgermline sequences.

In some embodiments, the nucleotide sequence and/or amino acid sequenceof the introduced transcribed variable region may be human, i.e., maycontain the nucleotide and/or amino acid sequence of a human antibody orgermline sequence. In these embodiments, both the CDRs and the frameworkmay be human. In other embodiments, the nucleotide sequence and/or aminoacid sequence of the introduced transcribed variable region is not humanand may instead be at least 80% identical, at least 90% identical, atleast 95% or more identical to a human sequence. For example, relativeto a human sequence, the introduced transcribed variable region maycontain one or more nucleotide or amino acid substitution. In particularembodiments, the nucleotide sequence of the introduced transcribedvariable region may be at least 80% identical, at least 90% identical,at least 95% or more identical to the variable regions shown in FIGS. 11and 12. In one embodiment, the framework sequence used contains one,two, three, four or five or more substitutions relative the frameworksequence shown in FIGS. 11 and 12.

In particular embodiments, part of the light chain locus that includesthe constant domain-encoding region, part of an intron, and the 3′UTR ofthe functional gene may be endogenous to the animal and the remainder ofthe light chain locus, including the variable regions of the functionalgene, the remainder of the intron and the pseudogenes may be exogenousto the animal, i.e., made recombinantly and introduced into the animalproximal to the constant domain, part intron and 3′ UTR in such a waythat a functional light chain gene is produced and the pseudogenes arecapable of donating sequence to the functional light chain gene by geneconversion. In certain cases the light chain locus of the animal maycontain, in operable linkage: an intron region, a constantdomain-encoding region and a 3′ untranslated region; where the intronregion, the constant domain-encoding region and the 3′ untranslatedregion are endogenous to the genome of the transgenic animal and aplurality of pseudogene light chain variable regions, where theplurality of pseudogene light chain variable regions are exogenous tothe genome of the transgenic animal. Alternatively, the constant domainencoding region could also be exogenous to the genome of the transgenicanimal.

Likewise, part of the heavy chain locus, including the constant region,part of an intron region and the 3′UTR of the functional gene, may beendogenous to the animal and the remainder of the heavy chain locus,including the variable domains of the functional gene, the remainder ofthe intron and the pseudogenes may be exogenous to the animal, i.e.,made recombinantly and introduced into the animal proximal to theconstant domain, part intron and 3′ UTR in such a way that a functionalgene is produced and the pseudogenes are capable of donating sequence tothe functional gene by gene conversion. In certain cases the heavy chainlocus of the animal may contain, in operable linkage: an intron region,a constant domain-encoding region and a 3′ untranslated region, wherethe intron region, the constant domain-encoding region and the 3′untranslated region are endogenous to the genome of the transgenicanimal, and a plurality of pseudogene heavy chain variable regions,where the plurality of pseudogene heavy chain variable regions areexogenous to the genome of the transgenic animal.

In certain embodiments, an antibody produced by a subject transgenicanimal may contain an endogenous constant domain and variable domainsthat are exogenous to the animal. Since an endogenous constant regionmay be employed in these embodiments, the antibody may still undergoclass switching and affinity maturation, which allows the animal toundergo normal immune system development, and mount normal immuneresponses. In specific embodiments transgenic chickens have threeendogenous constant regions in the heavy chain locus encoding IgM, IgYand IgA. During the early stages of B cell development, B cells expressIgM. As affinity maturation proceeds, class switching converts theconstant region into IgY or IgA. IgY provides humoral immunity to bothadults and neonatal chicks which receive about 200 mg of IgY via areserve deposited into egg yolk. IgA is found primarily in lymphoidtissues (eg. the spleen, Peyer's patches and Harderian glands) and inthe oviduct.

While, as noted above, the encoded framework regions of the variableregions of both the pseudogenes and the functional gene of the lightchain locus may be identical to one another, the CDR regions encoded bythe variable regions in each of the pseudogenes are different to oneanother (i.e., each of the plurality of pseudogenes encodes a CDR1region that is different to the amino acid sequences of all the otherCDR1 regions, each of the plurality of pseudogenes encodes a CDR2 regionthat is different to the amino acid sequences of all the other CDR2regions, and each of the plurality of pseudogenes encodes a CDR3 regionthat is different to the amino acid sequences of all the other CDR3regions). Likewise for the heavy chain locus, the CDR regions encoded bythe variable regions in each of the pseudogenes are different to oneanother.

In certain cases, the CDR regions encoded by the light chain variabledomain, and/or the heavy chain variable domain may be composed of only 2to 5 (i.e., 2, 3, 4, or 5) different amino acid residues, where, in thiscontext, the term “composed of” is intended to mean that each individualamino acid position within a CDR is occupied by a single amino acidresidue independently chosen from a group of 2 to 5 amino acid residues.Examples of CDRs that are composed of 2-5 amino acids are described inthe Examples section of this disclosure. In certain embodiments, atleast one of the 2 to 5 amino acids is a bulky amino acid such as atyrosine or tryptophan residue, and at least one of said 2 to 5 aminoacids is a small amino acid residue such as an alanine, glycine orserine residue.

CDRs may vary in length. In certain embodiments, the heavy chain CDR1may be in the range of 6 to 12 amino acid residues in length, the heavychain CDR2 may be in the range of 4 to 12 amino acid residues in length,the heavy chain CDR3 may be in the range of 3 to 25 amino acid residuesin length, the light chain CDR1 may be in the range of 4 to 14 aminoacid residues in length, the light chain CDR2 may be in the range of 2to 10 amino acid residues in length, the light chain CDR3 may be in therange of 5 to 11 amino acid residues in length, although antibodieshaving CDRs of lengths outside of these ranges are envisioned.

With the exception of a relatively small number of amino acids arisingas a result of mutations that occur independently of gene conversionduring affinity maturation (which occur in, e.g., less than 10%, lessthan 5%, less then 3%, or less than 1% of the amino acids), theresultant antibodies produced by the transgenic animal may have lightand/or heavy chain CDRs that are solely composed of the 2 to 5 differentamino acids. In exemplary embodiments, the CDRs are composed of 25% to75% (e.g., 40% to 60%) bulky amino acids selected from tyrosine andtryptophan, and 25% to 75% (e.g., 40% to 60%) small amino acids selectedfrom alanine, glycine and serine, with the remainder (i.e., less than10%, less than 5%, less then 3%, or less than 1% of the amino acids),being any of the other naturally occurring amino acids. The particularorder of the amino acids in each CDRs of the pseudogenes may be randomlygenerated.

The number of introduced pseudogene variable regions present at thelight and/or heavy chain locus may vary and, in particular embodiments,may be in the range of 5-30, e.g., 10 to 25. In particular embodiments,at least one (e.g., at least 2, at least 3, at least 5, at least 10 ormore) of the plurality of pseudogene light chain variable regions may bein reverse orientation relative to the transcribed light chain variableregion. Likewise, in particular embodiments, at least one (e.g., atleast 2, at least 3, at least 5, at least 10 or more) of the pluralityof pseudogene heavy chain variable regions may be in reverse orientationrelative to the heavy chain transcribed variable region. In particularembodiments, the plurality of pseudogene variable regions are not inalternating orientations, and in certain cases may (as illustrated inFIG. 8) rather contain a series of at least 5 or at least 10 adjacentpseudogene regions that are in opposite orientation relative to thetranscribed variable region. In one embodiment, the pseudogene regionthat is most distal from the transcribed variable region is in the sameorientation as the transcribed variable region, and the pseudogeneregions between the most distal region and the transcribed variableregion are in the reverse orientation relative to the transcribedvariable region.

The above-described transgenic animal may be made by replacing theendogenous variable regions in an endogenous immunoglobulin light chainlocus and/or heavy chain locus of animal with a plurality of pseudogenelight chain variable regions constructed recombinantly. Methods forproducing transgenic animals that use gene conversion to generate anantibody repertoire are known (see, e.g., Sayegh, Vet. Immunol.Immunopathol. 1999 72:31-7 and Kamihira, Adv. Biochem. Eng. Biotechnol.2004 91: 171-89 for birds, and Bosze, Transgenic Res. 2003 12:541-53 andFan, Pathol. Int. 1999 49: 583-94 for rabbits and Salamone J.Biotechnol. 2006 124: 469-72 for cow), as is the structure and/orsequence of the germline immunoglobulin heavy and light chain loci ofmany of those species (e.g., Butler Rev Sci Tech 1998 17:43-70 andRatcliffe Dev Comp Immunol 2006 30: 101-118), the above-described animalmay be made by routine methods given this disclosure.

A method of making a transgenic animal is provided. In certainembodiments, the method comprises: replacing the variable regions in theendogenous immunoglobulin light chain locus of a suitable animal with anucleic acid construct comprising: a) a light chain variable regionencoding: i. light chain CDR1, CDR2 and CDR3 regions that are composedof 2 to 5 different amino acids; and ii. light chain framework regions;and b) a plurality of pseudogene light chain variable regions eachencoding: i. light chain CDR1, CDR2 and CDR3 regions that are composedof the 2 to 5 different amino acids; and ii. light chain frameworkregions that are identical to the corresponding framework regionsencoded by the light chain variable region. Upon integration of theconstruct, the light chain variable region becomes the transcribedvariable region of the functional immunoglobulin locus of the transgenicanimal, and the pseudogene variable regions alter the sequence of thetranscribed V region by gene conversion. In particular embodiments, theengineered locus is designed to fully replace the endogenous V region,including pseudo-V's, the transcribed V, as well as the D and J genesegments. However, non-coding sequences (introns) may be retained inendogenous configuration in order to preserve endogenous regulatoryelements that may be contained within.

Likewise, the method may comprise: replacing the variable regions in theendogenous immunoglobulin heavy chain locus of the animal with a) aheavy chain variable region encoding: i. light chain CDR1, CDR2 and CDR3regions that are composed of the 2 to 5 different amino acids; and ii.heavy chain framework regions; and b) a plurality of pseudogene heavychain variable regions each encoding: i. heavy chain CDR1, CDR2 and CDR3regions that are composed of the 2 to 5 different amino acids; and ii.heavy chain framework regions that are identical to the correspondingframework regions encoded by the heavy chain variable region. Uponintegration of the construct, the variable region becomes thetranscribed variable region of the functional immunoglobulin locus ofthe transgenic animal, and the pseudogene V regions alter the sequenceof the transcribed variable region by gene conversion. Gene conversionmay result in the contribution of small (eg 1-10 nucleotides), moderate(10-30 nucleotides), or large (>30 nucleotides) segments of DNA from oneor more of the donor pseudogenes to the transcribed V region. Geneconversion can transpire over many iterations, so multiple pseudo-V'smay contribute sequence to the actively expressed V gene. Since theprocess of gene conversion is highly variable in terms of whichpseudogenes are selected, and the extent to which each is utilized in agiven lymphocyte, a large and diverse antibody repertoire will result inthe transgenic animal.

As would be readily apparent, the method may include first deleting aregion containing the variable regions in the endogenous immunoglobulinlight chain locus of the animal (including the transcribed variableregion and the pseudogene variable regions, and all sequences inbetween) to leave, e.g., a constant region sequence and part of theintron between the constant region sequence and the transcribed variableregion; and then adding the transcribed light chain variable region, theremainder of the intron, and the plurality of pseudogene light chainvariable regions to the locus of the mammal.

In particular embodiments and as schematically illustrated in FIGS. 5and 7, at least the variable region of the endogenous functionalimmunoglobulin gene of the transgenic animal may be replaced by anucleic acid construct containing a plurality of pseudogene variableregions and a transcribed variable region, without replacing theendogenous pseudogene variable regions of said transgenic animal. Assuch, the resultant immunoglobulin locus (which may be the heavy orlight chain locus) may contain an array of endogenous pseudogenes inaddition to an array of introduced pseudogenes upstream of a transcribedvariable region.

Once a subject transgenic animal is made, antibodies against an antigencan be readily obtained by immunizing the animal with the antigen. Avariety of antigens can be used to immunize a transgenic host animal.Such antigens include, microorganism, e.g. viruses and unicellularorganisms (such as bacteria and fungi), alive, attenuated or dead,fragments of the microorganisms, or antigenic molecules isolated fromthe microorganisms.

In certain embodiments, the animal may be immunized with: GD2, EGF-R,CEA, CD52, CD20, Lym-1, CD6, complement activating receptor (CAR),EGP40, VEGF, tumor-associated glycoprotein TAG-72 AFP(alpha-fetoprotein), BLyS (TNF and APOL-related ligand), CA125(carcinoma antigen 125), CEA (carcinoembrionic antigen), CD2 (T-cellsurface antigen), CD3 (heteromultimer associated with the TCR), CD4,CD11a (integrin alpha-L), CD14 (monocyte differentiation antigen), CD20,CD22 (B-cell receptor), CD23 (low affinity IgE receptor), CD25 (IL-2receptor alpha chain), CD30 (cytokine receptor), CD33 (myeloid cellsurface antigen), CD40 (tumor necrosis factor receptor), CD44v6(mediates adhesion of leukocytes), CD52 (CAMPATH-1), CD80 (costimulatorfor CD28 and CTLA-4), complement component C5, CTLA, EGFR, eotaxin(cytokine A11), HER2/neu, HER3, HLA-DR, HLA-DR10, HLA ClassII, IgE,GPiib/iiia (integrin), Integrin aVß3, Integrins a4ß1 and a4ß7, Integrinß2, IFN-gamma, IL-1ß, IL-4, IL-5, IL-6R (IL6 receptor), IL-12, IL-15,KDR (VEGFR-2), lewisy, mesothelin, MUC1, MUC18, NCAM (neural celladhesion molecule), oncofetal fibronectin, PDGFßR (Beta platelet-derivedgrowth factor receptor), PMSA, renal carcinoma antigen G250, RSV,E-Selectin, TGFbeta1, TGFbeta2, TNFα, DR4, DR5, DR6, VAP-1 (vascularadhesion protein 1) or VEGF, or the like in order to produce atherapeutic antibody.

The antigens can be administered to a transgenic host animal in anyconvenient manner, with or without an adjuvant, and can be administeredin accordance with a predetermined schedule.

After immunization, serum or milk from the immunized transgenic animalscan be fractionated for the purification of pharmaceutical gradepolyclonal antibodies specific for the antigen. In the case oftransgenic birds, antibodies can also be made by fractionating eggyolks. A concentrated, purified immunoglobulin fraction may be obtainedby chromatography (affinity, ionic exchange, gel filtration, etc.),selective precipitation with salts such as ammonium sulfate, organicsolvents such as ethanol, or polymers such as polyethyleneglycol.

For making a monoclonal antibody, antibody-producing cells, e.g., spleencells, may isolated from the immunized transgenic animal and used eitherin cell fusion with transformed cell lines for the production ofhybridomas, or cDNAs encoding antibodies are cloned by standardmolecular biology techniques and expressed in transfected cells. Theprocedures for making monoclonal antibodies are well established in theart. See, e.g., European Patent Application 0 583 980 A1, U.S. Pat. No.4,977,081, WO 97/16537, and EP 0 491 057 B1, the disclosures of whichare incorporated herein by reference. In vitro production of monoclonalantibodies from cloned cDNA molecules has been described byAndris-Widhopf et al., J Immunol Methods 242:159 (2000), and by Burton,Immunotechnology 1:87 (1995), the disclosures of which are incorporatedherein by reference.

As such, in addition to the transgenic animal, a method comprisingimmunizing the transgenic animal with an antigen and obtaining from thetransgenic animal an antibody that specifically binds to the antigen isalso provided. The method may include making hybridomas using cells ofthe transgenic animal; and screening the hybridomas to identify ahybridoma that produces an antibody that specifically binds to theantigen.

If the antibody does not already contain human framework regions, themethod may further include humanizing the antibody, which method mayinclude swapping the constant domain of the antibody with a humanconstant domain to make a chimeric antibody, as well as in certain caseshumanizing the variable domains of the antibody by e.g., CDR grafting orresurfacing etc. Humanization can be done following the method of Winter(Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323(1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J.Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901(1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992);Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat. Nos. 5,723,323,5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323,5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101,5,585,089, 5,225,539; 4,816,567, PCT/US98/16280, US96/18978, US91/09630,US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443,WO90/14424, WO90/14430, EP 229246, each entirely incorporated herein byreference, including references cited therein.

Antibody Compositions

Antibody compositions are provided. An antibody may minimally have theCDRs of an antibody produced b (i.e., light chain CDR1, CDR2 and CDR3and/or heavy chain CDR1, CDR2 and CDR3 regions of an antibody producedby a subject animal) and in the one embodiment will contain the entirevariable domains (i.e., CDR plus framework) of an antibody produced bythe subject animal. Such an antibody composition may contain polyclonalantisera or a monoclonal antibody that specifically binds to an antigen,methods for the production of which are known and described above.

Except for a relatively small number of amino acids that have resultedfrom non-gene conversion based amino acid changes to the variable domainin the functional gene during affinity maturation (i.e., which occur inless than 10%, less than 5, less than 3%, or less then 1% of the aminoacids), the CDRs of the light and/or heavy chain of a subject antibodyare composed the 2-5 amino acids encoded by locus described above.Likewise, the framework region is comprised of the predeterminedsequence known to have desirable attributes such as monomeric form, easeof manufacturing, high solubility, and thermodynamic stability.

As noted above, the heavy and light chains variable domains of theantibody are naturally paired by the immune system of the animal. Suchantibodies may, in certain case, be post-translationally modified (e.g.,glycosylated) by the host cell and may have a glycosylation pattern andcomposition characteristic of the species of transgenic animal.

In certain embodiments, an antibody produced by the transgenic animal isprovided, where the antibody comprises: a constant domain linked to avariable domain, wherein the variable domain comprises: a) a light chainvariable domain comprising: i. light chain CDR1, CDR2 and CDR3 regionsthat are composed of 2 to 5 different amino acids; and ii. light chainframework regions; and a) a heavy chain variable domain comprising: i.heavy chain CDR1, CDR2 and CDR3 regions that are composed of the 2 to 5different amino acids; and ii. heavy chain framework regions.

In particular embodiments, the resultant antibody may have a frameworkthat is at least 80% (e.g., at least 90%, at least 95% or more)identical to the framework of the antibody shown in FIGS. 11 and 12.

Methods of Screening

The antibodies produced by the subject transgenic animal may be screenedto identify an antibody of interest. In general, this method involvesproducing a plurality of hybrid cells producing monoclonal antibodiesusing the method described above, and screening the plurality ofmonoclonal antibodies using one or a combination of a variety of assays.In general, these assays are functional assays, and may be grouped asfollows: assays that detect an antibody's binding affinity orspecificity, and assays that detect the ability of an antibody toinhibit a process.

A monoclonal antibody identified as having a specific binding activitywith an antigen, or an inhibitory activity is termed a monoclonalantibody of interest.

Binding Assays

In these assays, antibodies are tested for their ability to bindspecifically to a substrate. The term “specifically” in the context ofantibody binding, refers to high avidity and/or high affinity binding ofan antibody to a specific antigen i.e., a polypeptide, or epitope. Inmany embodiments, the specific antigen is an antigen (or a fragment orsubfraction of an antigen) used to immunize the animal host from whichthe antibody-producing cells were isolated. Antibody specificallybinding an antigen or fragment thereof is stronger than binding of thesame antibody to other antigens. Antibodies which bind specifically to apolypeptide may be capable of binding other polypeptides at a weak, yetdetectable, level (e.g., 10% or less of the binding shown to thepolypeptide of interest). Such weak binding, or background binding, isreadily discernible from the specific antibody binding to a subjectpolypeptide, e.g. by use of appropriate controls. In general, specificantibodies bind to an antigen with a binding affinity of 10⁻⁷ M or more,e.g., 10⁻⁸ M or more (e.g., 10⁻⁹ M, 10⁻¹⁰, 10⁻¹¹, etc.). In general, anantibody with a binding affinity of 10⁻⁶ M or less is not useful in thatit will not bind an antigen at a detectable level using conventionalmethodology currently used.

Typically, in performing a screening assay, antibody samples produced bya library of antibody producing host cells are deposited onto a solidsupport in a way that each antibody can be identified, e.g. with a platenumber and position on the plate, or another identifier that will allowthe identification of the host cell culture that produced the antibody.

The antibodies of the invention may be screened for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, and protein A immunoassays, to name but a few. Such assaysare routine and well known in the art (see, e.g., Ausubel et al, eds,1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,Inc., New York, which is incorporated by reference herein in itsentirety). Exemplary immunoassays are described briefly below (but arenot intended by way of limitation).

Immunoprecipitation protocols generally involve lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4 .degree. C., adding protein Aand/or protein G sepharose beads to the cell lysate, incubating forabout an hour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads).

Western blot analysis generally involves preparation of protein samplesfollowed by electrophoresis of the protein samples in a polyacrylamidegel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), and transfer of the separated protein samples from thepolyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon.Following transfer, the membrane is blocked in blocking solution (e.g.,PBS with 3% BSA or non-fat milk), washed in washing buffer (e.g.,PBS-Tween 20), and incubated with primary antibody (the antibody ofinterest) diluted in blocking buffer. After this incubation, themembrane is washed in washing buffer, incubated with a secondaryantibody (which recognizes the primary antibody, e.g., an anti-humanantibody) conjugated to an enzymatic substrate (e.g., horseradishperoxidase or alkaline phosphatase) or radioactive molecule (e.g., 32Por 125I), and after a further wash, the presence of the antigen may bedetected. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise.

ELISAs involve preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., 3H or 125I)in the presence of increasing amounts of an unlabeled second antibody.

Antibodies of the invention may be screened using immunocytochemistymethods on cells (e.g., mammalian cells, such as CHO cells) transfectedwith a vector enabling the expression of an antigen or with vector aloneusing techniques commonly known in the art. Antibodies that bind antigentransfected cells, but not vector-only transfected cells, are antigenspecific.

In certain embodiments, however, the assay is an antigen capture assay,and an array or microarray of antibodies may be employed for thispurpose. Methods for making and using microarrays of polypeptides areknown in the art (see e.g. U.S. Pat. Nos. 6,372,483, 6,352,842,6,346,416 and 6,242,266).

Inhibitor Assays

In certain embodiments, the assay measures the specific inhibition of anantibody to an interaction between a first compound and a secondcompound (e.g. two biopolymeric compounds) or specifically inhibits areaction (e.g. an enzymatic reaction). In the interaction inhibitionassay, one interaction substrate, usually a biopolymeric compound suchas a protein e.g. a receptor, may be bound to a solid support in areaction vessel. Antibody is added to the reaction vessel followed by adetectable binding partner for the substrate, usually a biopolymericcompound such as a protein e.g. a radiolabeled ligand for the receptor.After washing the vessel, interaction inhibition may be measured bydetermining the amount of detectable binding partner present in thevessel. Interaction inhibition occurs when binding of the bindingpartner is reduced greater than about 20%, greater than about 50%,greater than about 70%, greater than about 80%, or greater than about90% or 95% or more, as compared to a control assay that does not containantibody.

In the reaction inhibition assay, an enzyme may be bound to a solidsupport in a reaction vessel. Antibody is usually added to the reactionvessel followed by a substrate for the enzyme. In many embodiments, theproducts of the reaction between the enzyme and the substrate aredetectable, and, after a certain time, the reaction is usually stopped.After the reaction has been stopped, reaction inhibition may be measuredby determining the level of detectable reaction product present in thevessel. Reaction inhibition occurs when the rate of the reaction isreduced greater than about 20%, greater than about 50%, greater thanabout 70%, greater than about 80%, or greater than about 90% or 95% ormore, as compared to a control assay that does not contain antibody.

In Vivo Assays

In certain embodiments the monoclonal antibodies are tested in vivo. Ingeneral, the method involves administering a subject monoclonal antibodyto an animal model for a disease or condition and determining the effectof the monoclonal antibody on the disease or condition of the modelanimal. In vivo assays of the invention include controls, where suitablecontrols include a sample in the absence of the monoclonal antibody.Generally a plurality of assay mixtures is run in parallel withdifferent antibody concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e. at zero concentration or below thelevel of detection.

A monoclonal antibody of interest is one that modulates, i.e., reducesor increases a symptom of the animal model disease or condition by atleast about 10%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 80%, at least about 90%, ormore, when compared to a control in the absence of the antibody. Ingeneral, a monoclonal antibody of interest will cause a subject animalto be more similar to an equivalent animal that is not suffering fromthe disease or condition. Monoclonal antibodies that have therapeuticvalue that have been identified using the methods and compositions ofthe invention are termed “therapeutic” antibodies.

Since a hybrid cell expressing an antibody of interest containsimmunoglobulin heavy and light chain-encoding nucleic acids, the nucleicacids encoding the monoclonal antibody of interest may be identified ifthe host cell expressing the monoclonal antibody of interest isidentified. As such, the subject nucleic acids may be identified by avariety of methods known to one of skill in the art. Similar methods areused to identify host cell cultures in monoclonal antibody productionusing hybridoma technology (Harlow et al., Antibodies: A LaboratoryManual, First Edition (1988) Cold spring Harbor, N.Y.).

For example, upon identifying a monoclonal antibody of interest, thehost cell expressing the antibody of interest may be identified using a“look-up” table which lists, for every antibody sample, thecorresponding host cell culture. In certain other embodiments, a look-uptable containing antibody library sample identifiers, correspondingexpression cassette library sample identifiers and/or host cellidentifiers may be used to identify the subject nucleic acids.

Once identified, the nucleic acids encoding a monoclonal antibody ofinterest may be recovered, characterized and manipulated usingtechniques familiar to one of skill in the art (Ausubel, et al, ShortProtocols in Molecular Biology, 3rd ed., Wiley & Sons, (1995) andSambrook, et al, Molecular Cloning: A Laboratory Manual, Third Edition,(2001) Cold Spring Harbor, N.Y.).

Antibody Expression

Also provided are several methods of producing a monoclonal antibody ofinterest. In general these methods involve incubating a host cellcontaining a nucleic acid encoding a monoclonal antibody of interestunder conditions sufficient for production of the antibody.

In some embodiments, the methods of producing a monoclonal antibody ofinterest involve transferring identified expression cassettes for amonoclonal antibody of interest into a suitable vector, and transferringthe recombinant vector into a host cell to provide for expression of themonoclonal antibody. In some embodiments, the subject methods involvetransferring at least the variable domain-encoding sequences from theidentified heavy and light chains into vectors suitable for theirexpression in immunoglobulin heavy and light chains. Suitable constantdomain-encoding sequences and/or other antibody domain-encodingsequences may be added to the variable domain-encoding sequences at thispoint. These nucleic acid modifications may also allow for humanizationof the subject antibody.

The subject monoclonal antibodies can be produced by any method known inthe art for the synthesis of antibodies, in particular, by recombinantexpression techniques.

Recombinant expression of a subject monoclonal antibody, or fragment,derivative or analog thereof, usually requires construction of anexpression vector containing a polynucleotide that encodes the antibody.Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques andsynthetic techniques. As such, the invention provides vectors comprisinga nucleotide sequence encoding an antibody molecule of the invention.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured to produce asubject antibody. In most embodiments, vectors encoding both the heavyand light chains are co-expressed in the host cell to provide forexpression of the entire immunoglobulin molecule.

A variety of host-expression vector systems may be utilized to express asubject monoclonal antibody. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3cells etc.) harboring recombinant expression constructs containingpromoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). In manyembodiments, bacterial cells such as Escherichia coli, and eukaryoticcells are used for the expression of entire recombinant antibodymolecules. For example, mammalian cells such as Chinese hamster ovarycells (CHO), in conjunction with a vector such as the major intermediateearly gene promoter element from human cytomegalovirus is an effectiveexpression system for antibodies (Foecking et al., Gene 45:101 (1986);Cockett et al., Bio/Technology 8:2 (1990)).

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the antibody molecule beingexpressed. For example, when a large quantity of such a protein is to beproduced, for the generation of pharmaceutical compositions of anantibody molecule, vectors which direct the expression of high levels offusion protein products that are readily purified may be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibodycoding sequence may be ligated individually into the vector in framewith the lac Z coding region so that a fusion protein is produced; pINvectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); VanHeeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like.pGEX vectors may also be used to express foreign polypeptides as fusionproteins with glutathione 5-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption and binding to matrix glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express antibodies. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized to express a subject antibody. In cases where an adenovirusis used as an expression vector, the antibody coding sequence ofinterest may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing the antibody molecule ininfected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA81:355-359 (1984)). The efficiency of expression may be enhanced by theinclusion of appropriate transcription enhancer elements, transcriptionterminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544(1987)).

For long-term, high-yield production of recombinant antibodies, stableexpression may be used. For example, cell lines, which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with immunoglobulin expression cassettes and a selectablemarker. Following the introduction of the foreign DNA, engineered cellsmay be allowed to grow for 1-2 days in an enriched media, and then areswitched to a selective media. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into a chromosome and grow to form foci which inturn can be cloned and expanded into cell lines. Such engineered celllines may be particularly useful in screening and evaluation ofcompounds that interact directly or indirectly with the antibodymolecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharnacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); TIB TECH 11(5):155-215 (1993)); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, N Y (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, N Y(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, N Y (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain different selectable markers and origins ofreplication, which enable equal expression of heavy and light chainpolypeptides. Alternatively, a single vector may be used which encodes,and is capable of expressing, both heavy and light chain polypeptides.

Once an antibody molecule of the invention has been produced, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In many embodiments, antibodies are secretedfrom the cell into culture medium and harvested from the culture medium.

Utility

Also provided is a method for modulating or treating at least oneantigen-related disease, in a cell, tissue, organ, animal, or patient,as known in the art or as described herein, using at least one antibodyof the present invention, e.g., administering or contacting the cell,tissue, organ, animal, or patient with a therapeutic effective amount ofantibody. The present invention also provides a method for modulating ortreating at least one antigen related disease, in a cell, tissue, organ,animal, or patient including, but not limited to, at least one ofobesity, an immune related disease, a cardiovascular disease, aninfectious disease, a malignant disease or a neurologic disease.

Typically, treatment of pathologic conditions is effected byadministering an effective amount or dosage of at least one antibodycomposition that total, on average, a range from at least about 0.01 to500 milligrams of at least one antibody per kilogram of patient perdose, and, preferably, from at least about 0.1 to 100 milligramsantibody/kilogram of patient per single or multiple administration,depending upon the specific activity of the active agent contained inthe composition. Alternatively, the effective serum concentration cancomprise 0.1-5000 ng/ml serum concentration per single or multipleadministration. Suitable dosages are known to medical practitioners andwill, of course, depend upon the particular disease state, specificactivity of the composition being administered, and the particularpatient undergoing treatment. In some instances, to achieve the desiredtherapeutic amount, it can be necessary to provide for repeatedadministration, i.e., repeated individual administrations of aparticular monitored or metered dose, where the individualadministrations are repeated until the desired daily dose or effect isachieved.

A subject antibody can, in certain embodiments also be used indiagnostics where the antibody is conjugated to a detectable markers orused as primary antibodies with secondary antibodies that are conjugatedto detectable markers. Detectable markers, include radioactive andnon-radioactive labels and are well-known to those with skill in theart. Common non-radioactive labels include detectable enzymes such ashorseradish peroxidase, alkaline phosphatase and fluorescent molecules.Fluorescent molecules absorb light at one wavelength and emit it atanother, thus allowing visualization with, e.g., a fluorescentmicroscope. Spectrophotometers, fluorescence microscopes, fluorescentplate readers and flow sorters are well-known and are often used todetect specific molecules which have been made fluorescent by couplingthem covalently to a fluorescent dye. Fluorochromes such as greenfluorescent protein, red shifted mutants of green fluorescent protein,amino coumarin acetic acid (AMCA), fluorescein isothiocyanate (FITC),tetramethylchodamine isothiocyanate (TRITC), Texas Red, Cy3.0 and Cy5.0are examples of useful labels.

The molecules can be used in cell isolation strategies such asfluorescence-activated cell sorting (FACS) if fluorescent markers areused, In fluorescence-activated cell sorting, cells tagged withfluorescent molecules are sorted electronically on a flow cytometer suchas a Becton-Dickinson (San Jose, Calif.) FACS IV cytometer or equivalentinstrument. The fluorescent molecules are antibodies that recognizespecific cell surface antigens. The antibodies are conjugated tofluorescent markers such as fluorescein isothiocyanate (FITC) orPhycoerythrin (PE).

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain embodiments and aspects of the present invention andare not to be construed as limiting the scope thereof.

Example 1 Summary

Briefly, a chicken is engineered to produce antibody containing a humanframework with biophysical properties and that is easily manufacturedand has optimal pharmacological properties. The immunoglobulin genes ofthe engineered chicken will have an array of 20 synthetic pseudogeneswith the identical framework region and CDRs composed of randomsequences of serine, tyrosine, alanine and aspartate to generateantigen-specific, high-affinity antibodies. This line of chickens willbe immunized and monoclonal antibodies will be recovered.

Gene conversion of an array of VL pseudogenes where all pseudogenes havean identical framework region and the CDRs are composed of random arraysof serine, tyrosine, alanine and aspartate will be demonstrated usingDT40 cells from a virally transformed chicken pre-B cell line thatcontinues to diversify the light chain by gene conversion in vitro.Furthermore, DT40 cells undergo high rates of homologous recombinationwhich provides a straightforward route for replacement of the chickenfunctional variable region with a recombinant variable region.

Knock-in targeting vectors to replace the array of chicken light chain Vregions in DT40 cells with a synthetic array derived from a single humanframework region and CDRs comprised of serine, tyrosine, alanine andaspartate will be created. Gene conversion of the synthetic CDRs in asingle human framework will be demonstrated in DT40 cells.

An array of synthetic human V regions will be inserted into the chickenIgL and IgH loci of primordial germ cells (PGCs). The geneticallymodified PGCs will be used to create a line of birds from which humanantibodies can be obtained following immunization. These birds will bethe first transgenic animals yielding engineered human antibodies withpredictable manufacturing attributes and pharmacological properties.

The V_(K)3 framework sequence will be used because it has the highestsolubility, exists as a monomer, and is thermodynamically stable. TheV_(H)3 framework sequence will be used for the same reasons and becausethe V_(H)3 framework has been shown to be well expressed.

Example 2 Functional V and the Pseudogene Array

A functional V (i.e., V region) was obtained by extracting a consensussequence for the framework, CDR1 and CDR2 of the human V_(K)3 sequenceslisted the VBase database. Since no consensus can be derived for CDR3 ofV_(K)3, the humIGKV096 sequence from VBase was used; this V_(K)3sequence was confirmed in genomic DNA as well as in productiverearrangements. The pseudogenes were designed to use the consensussequence of V_(K)3 as the framework region and random arrays of tyrosine(Y), serine (S), and tryptophan (W) in CDR1, CDR2 and CDR3. Thesesequences are shown below in Table 1.

TABLE 1  Sequence of tyrosine (Y), serine (S), and trypto-phan (W) in CDR1, CDR2 and CDR3 in the functionalV_(κ)3 derived gene and in the pseudo-V (PSI) genes. CDR1 CDR2 CDR3 VK3QSVSSN GAS QQYNNW consensus (SEQ ID NO: 7) (SEQ ID NO: 28) PSI-1 YSSYSSYSS YSSYSS (SEQ ID NO: 8) (SEQ ID NO: 29) PSI-2 SYSSYS SYS SYSSYS(SEQ ID NO: 9) (SEQ ID NO: 30) PSI-3 SSYSSY SSY SSYSSY (SEQ ID NO: 10)(SEQ ID NO: 31) PSI-4 YSYSYS SSS YSYSYS (SEQ ID NO: 11) (SEQ ID NO: 32)PSI-5 SYSYSY SSS SYSYSY (SEQ ID NO: 12) (SEQ ID NO: 33) PSI-6 YSSSSY SSSYSSSSY (SEQ ID NO: 13) (SEQ ID NO: 34) PSI-7 YYSSSS YSS YYSSSS(SEQ ID NO: 14) (SEQ ID NO: 35) PSI-8 SYYSSS SYS SYYSSS (SEQ ID NO: 15)(SEQ ID NO: 36) PSI-9 SSYYSS SSY SSYYSS (SEQ ID NO: 16) (SEQ ID NO: 37)PSI-10 SSSYYS SSS SSSYYS (SEQ ID NO: 17) (SEQ ID NO: 38) PSI-11 SSSSYYSSS SSSSYY (SEQ ID NO: 18) (SEQ ID NO: 39) PSI-12 WSYSSS SSS WSYSSS(SEQ ID NO: 19) (SEQ ID NO: 40) PSI-13 SWSYSS YSS SWSYSS (SEQ ID NO: 20)(SEQ ID NO: 41) PSI-14 SSWSYS SYS SSWSYS (SEQ ID NO: 21) (SEQ ID NO: 42)PSI-15 SSSWSY SSY SSSWSY (SEQ ID NO: 22) (SEQ ID NO: 43) PSI-16 YSSSWSSSS YSSSWS (SEQ ID NO: 23) (SEQ ID NO: 44) PSI-17 SYSSSW SSS SYSSSW(SEQ ID NO: 24) (SEQ ID NO: 45) PSI-18 WYWYWY YSS WYWYWY (SEQ ID NO: 25)(SEQ ID NO: 46) PSI-19 YWYWYW SYS YWYWYW (SEQ ID NO: 26) (SEQ ID NO: 47)PSI-20 SSSSSS SSY SSSSSS (SEQ ID NO: 27) (SEQ ID NO: 48)

The CDR regions of the pseudogene array were constructed from only 3amino acids: tyrosine (Y), serine (S), and tryptophan (W) in proportionsof 40/50/10%, respectively. In this strategy, tyrosine and tryptophanare predicted to be the primary antigen-contact residues, while serineprovides appropriate spacing within the binding pocket. The array isdesigned to allow a Y or W to appear in any position of any CDR and tocollectively provide a sufficient number of alternatively orderedsequences to generate any possible sequence efficiently through geneconversion. This design assumes that diversity will be generated fromthe synthetic array using the same gene conversion process that is usedto generate a repertoire from the pseudogene array in wild-typechickens.

The performance of this array was tested in silico, by creating a panelof mock “antigen-selected” CDR sequences comprised of Y, S and Wresidues. The approximate composition of the array (listed in Table 1)was 40% tyrosine, 50% serine, and 10% tryptophan, a distribution that isthought to be optimal in vitro. The simulated amino acid sequence (SAAS)of CDR1 (or CDR3 since both have 6 positions) was created using a randomnumber generator and assigning a value of 0 to 0.4 to tyrosine, >0.4 to0.9 to serine and >0.9 to 1 to tryptophan. The output of this simulationfor CDR1 (or CDR3) is shown in Table 2.

TABLE 2 The simulated amino acid sequence (SAAS) of CDR1 (or CDR3) andthe gene conversion (GC) events needed to generate the predictedsequence from the pseudogene (PSI) array in Table 1. CDR Position # GC 12 3 4 5 6 events PSI used SAAS-1 S Y S S S S 2 p 2, 7 SAAS-2 S S S Y W S2 p 10, 16 SAAS-3 S S Y Y S Y 2 p 9, 6 SAAS-4 W S S Y Y W 3 p 12, 10, 17SAAS-5 S W Y W S S 3 p 13, 19, 7 SAAS-6 Y S W W S S 4 p 4, 14, 15, 12SAAS-7 S Y W Y S S 3 p 2, 14, 9 SAAS-8 S Y S Y Y S 2 p 5, 10 SAAS-9 S SY Y W Y 2 p 9, 18 SAAS-10 Y Y S Y Y Y 3 p 7, 5, 11 SAAS-11 Y Y Y Y Y S 3p 7, 9, 10 SAAS-12 Y W W W W Y 3 p 18, 19, 18 SAAS-13 W S Y S S Y 2 p12, 6 SAAS-14 W S S Y Y Y 2 p 12, 3 SAAS-15 S Y Y W W S 3 p 8, 15.16Average 2.6

The minimum number of gene conversions that would be required to achievethe simulated amino acid sequences in Table 2 and the PSI that could beused to create the SAAS was manually evaluated and are shown in thecolumn “GC events”. Generally we were able to create any SAAS with only2 or 3 gene conversion-like events, with an average of 2.6. Since thepublished estimates of gene conversion frequency ranges from 3-6independent events per V gene, the pseudogene array should be able toproduce sufficient sequence and functional diversity to generate highlyspecific clones to any antigenic sequence.

Example 3 Gene Targeting Constructs

Modification of the genome will be done in two steps. First a genetargeting vector will be introduced into the chicken DT-40 cells toremove the entire array of endogenous chicken light chain pseudo V genes(around 20 Kb sequences) as well as the chicken functional V region(including leader, VJ and corresponding intronic sequences). Theschematic design of the targeting vector is illustrated in FIG. 1.

Sequences upstream of the pseudo V25 will be used as the 5′ homologousregion and sequences downstream of J in the J-C intron can be used asthe 3′ homologous region to facilitate the targeting by homologousrecombination. A loxP flanked neoR cassette driven by the β-actinpromoter will be used for selection which can then be removed in thetargeted cells by cre-mediated recombination. In addition, an attP siteand a promoterless puroR (with a FRT sequence at 3′ end) will beincluded in the targeting vector (see description below). Thisfacilitates the easy integration of other arrays of synthetic Vs ifnecessary. The resulting targeted locus will have the entire chickenpseudo V and functional V array removed. In the second step, a syntheticV array (with both synthetic pseudo Vs and functional V_(K)3 shown asL-sVJ in FIG. 2) in a replacement vector with an attB site and apromoter (with a FRT sequence at 5′ end) will be inserted to thepre-targeted locus at the attP site via a phage phiC31integrase-mediated site-specific recombination (FIG. 2). The correctintegration event will bring the promoter and the promoterless puroRtogether to allow selection with puro. This will further enrich thecorrect integration at the pre-introduced attP site to almost 100%efficiency against integrations at pseudo attP sites present in thechicken genome. Again the promoter-puroR sequence can be removed via FRTif necessary.

The assembly of the synthetic V array is illustrated in FIG. 3 and FIG.4. Specifically, the In-Fusion technology developed by Clontech will beused to generate subfragments of synthetic Vs for downstream cloninginto the final vector. The In-Fusion technology can efficiently “fuse”DNA fragments with a 15-bp overlap of homologous sequence at the ends.The 15 bp homologous ends can be added to the end of gene-specific PCRprimers. This technology has been used to efficiently assemble four ormore pieces of DNA seamlessly. To reconstitute the synthetic pseudo Varray, 39 segments (20 synthetic pseudo Vs and 19 inter-pseudo Vsequences derived from endogenous chicken IgL sequences) spanning about25 Kb will be joined together. Synthetic pseudo V genes will be obtainedby gene synthesis. Inter-pseudo V sequences will be obtained by PCR fromBAC DNA. A BAC clone (CH261-29C12) containing the entire cIgL locus willbe obtained from the Children's Hospital Oakland Research Institute(CHORI). In the process 15-bp homologous ends will be added to the endsof each segment. Initially, 3 segments will be assembled together byIn-Fusion. Each segment contains a 15 bp sequence that is homologous(color-coded) to the adjacent segments or vectors. A modifiedpBluescript-based intermediate vector will be used as the linearizedvector DNA for In-Fusion. After the first round of In-Fusion, there are13 intermediate segments. These 13 segments will go through a secondround of In-Fusion to yield 5 final segments which will contain up to 9starting segments each.

Unique restriction sites will be added at the indicated locations of thefinal segments to facilitate the assembly of the entire synthetic pseudoV locus via conventional restriction site-mediated cloning. ApBeloBAC-based vector will be modified to include attB and the Promoter,and 7 unique restriction sites (RE-1 to RE-7) to allow the finalassembly of the synthetic V array. A DNA fragment containing theendogenous chicken sequence between pseudo V1-Leader and sequence ofleader-Synthetic human VJ framework (L-sVJ in FIG. 4) will be clonedinto the vector to reconstruct the entire VL locus.

Since the frequency of gene conversion can be sensitive to transcriptionof nearby loci we have designed the constructs with Lox or FRT sitesflanking the selectable marker to facilitate its removal. To excise thegene encoding resistance to neomycin, the clones will be transfectedwith a plasmid expressing Cre-recombinase (Vector Biolabs) and to excisethe gene encoding resistance to puromycin, and the clones will betransfected with a plasmid expressing Flipase (Invitrogen). Cells willthen be singly seeded to obtain clones without the selectable marker.Once these clones are apparent (usually in 5-7 days), we will useSouthern analysis to verify that the antibiotic resistance gene has beenexcised. We will then add 1.5 ng/ml trichostatin A to the cultures toenhance gene conversion. During the next 4 weeks, we will monitorreversion to the sIgM+ phenotype and assess the contribution of chickenpseudo V regions to the human V sequence.

Example 4 Culture and Transfection of DT40 Cells

DT40 cells are grown in DMEM supplemented with 10% tryptose phosphatebroth, 55 μM beta-mercaptoethanol, 2 mM L-glutamine, 10% FBS and 2%chicken serum. Cells are seeded at 2.5×10⁵ cells/ml and are split at 2to 3 day intervals. Care is taken to prevent the cultures from becomingtoo dense.

For transfection 5×10⁶ cells in logarithmic phase growth are collected,washed with PBS, pelleted and re-suspended in 100 μl Amaxa V-buffer.Five μg of linearized DNA is added, the suspension is transferred to acuvette and electroporated using an exponential decay pulse of 550V and25 μF. Immediately after electroporation the cells are put in 500 μl ofprewarmed medium in an 1.5 ml Eppendorf tube and placed at 37° C. After20 minutes, the cells are resuspended in 40 mls of medium and aliquotsof 1000 are deposited into four 96-well plates. The day aftertransfection an equal volume (1000) of medium containing the antibioticbeing used for selection (e.g puromycin in a 2× concentration of 1mg/ml) is added for a final puromycin concentration of 0.5 mg/ml. Within5 to 7 days, colonies grow to about 2 mm in diameter and are transferredto 24-well plates. Colonies are then expanded using the same cultureconditions that are described above.

Stably transfected clones will be analyzed by PCR to determine targetingand targeting will be confirmed in prospective clones by Southernanalysis. We will identify at least two clones by Southern analysis foreach of the genetic modifications.

Example 5 Evaluation of Gene Conversion Using the sIgM Reversion Assay

The rate of gene conversion will be monitored by an sIgM+ reversionassay (Yang e al., J. Exp. Med. 203: 2919-2928, 2006). In this system,the frameshift variant DT40-CL18, which has a single base insertion atposition 128 in the VL gene, prevents the light chain from pairing withendogenous heavy chain, and thus the cells are surface IgM− (Yang et al,2006). When the frameshift mutation is reverted due to a gene conversionevent, the cells become sIgM+, a phenotype that is readily identified byFluorescence Activated Cell Sorting (FACS). Four weeks after starting aculture from the DT40-CL18 frameshift variant, approximately 1.5% of thecells will be sIgM+. These clones are then recovered and the V regionssequenced to fully document diversification of the functional V.

In this application, the functional V_(K)3 gene is a consensus sequenceand therefore, the CDRs are composed of tryptophan, glycine, valine,glutamine and asparagine in addition to serine, alanine and tyrosine.Accumulation of the four amino acids in the synthetic pseudo Vs will bethe metric for gene conversion.

To use the sIgM reversion assay in this application, the humanfunctional V region will be engineered to contain an amber stop codon inCDR1. Hence, both wild type and mutant versions of the transgene will bealternatively inserted into the introduced attP site in the genome ofDT40 cells. The wild type version will provide evidence that thefunctional light chain is capable of pairing with the endogenous heavychain to reconstitute IgM expression. Since both heavy and light chainconstant regions are of chicken origin, it is likely that normal pairingwill occur, but there is a possibility of interference between the humanand chicken V regions. If that is the case, we will not be able to takeadvantage of the sIgM+ reversion assay and will rely exclusively onsequencing analysis for evaluation of gene conversion events. However,complete antibodies are assembled when murine variable regions arespliced onto chicken constant regions lending credence to oursupposition that chimeric human-chicken light chains will pair withchicken heavy chains. Furthermore, V_(K)3 is about 70% homologous tochicken functional Vs and this level of homology is expected tofacilitate productive pairing.

At the end of the four week culture period, the cells expressing themutant version of the transgene will be stained with a rabbitanti-chicken Ig (Sigma) and single sIgM+ cells will be sorted by FACSinto 96 well plates. These data will provide the first indication thatgene conversion has occurred and we anticipate that approximately 1.5%of the cells will be sIgM+(Yang et al, 2006). The cells will be grownfor 5-7 days and IgM mRNA will be prepared from each of the wells. Theresulting cDNA will be amplified with a 5′ leader peptide primer and a3′ CL primer. Both primers are of chicken origin and will thereforeamplify VL regardless of the extent of gene conversion. Amplicons willbe cloned into the TOPO TA cloning vector (Invitrogen). Plasmid DNA fromE. coli transformant colonies will be prepared from each well and thecloned insert will be sequenced. Sequences will be analyzed relative tothe original CDRs and the accumulation of tyrosine, serine andtryptophan will be used as an index of gene conversion.

Example 6 Knock-Out Vectors and Transfection of the Same in DT40 Cells

The data presented in the following examples show that human Ig lightchain transgene comprised of a single framework region and an array ofupstream human synthetic pseudogenes can be inserted into the chickenlight chain locus of the chicken B cell line DT40. Gene conversiondiversifies the synthetic CDRs in chicken B cells.

The replacement of the chicken light chain locus with human V regionswas done in two steps, as described above: knockout of the chIgL locusand placement of an attP site in the locus, followed by knock-in of thehuman V regions using integrase. The knockout vector was designed todelete the V, J and C regions (FIG. 5), leaving behind the chickenpseudogenes, which would not be predicted to interfere with geneconversion since sequence homology to the human V is low (only smallstretches of weak homology in framework region 2).

5′ and 3′ homology arms for the targeting vector were prepared by PCRamplification of genomic DNA and assembled with the puromycin, EGFP andpromoterless neo selectable cassette. The EGFP marker is useful foridentifying and tracking transfected cells and colonies, especially inthe case of gonocytes because the feeder layer that the cells are grownon sometimes makes visualization of small colonies difficult. Inaddition, EGFP facilitates the screening for germline transmission ofknockout or knock-in gonocytes by shining UV light onto chicks andassessing green fluorescence. The puromycin gene is used for selectionof the knockout clones, and the promoterless neo gene will be used laterfor selection of integrase-mediated insertion of the array ofpseudogenes. An attP site was placed in front of the neo gene forrecombination by ΦC31 integrase. loxP sites were included for laterremoval of the selectable markers by Cre recombinase.

The knockout vector transfected into wild type DT40 cells,puromycin-resistant clones were selected and clones that had integratedthe vector by homologous recombination were screened for, therebyknocking out the light chain. Of the two alleles of the light chain genein DT40, one is in germline configuration and is not expressed, whereasthe other has undergone VJ rearrangement to express the light chaingene. The rearranged allele may be knocked out because it eliminatesexpression of the endogenous light chain, leading to surfaceIgM-negative cells and simplifying downstream analysis. The germlineallele cannot rearrange because RAG-1 is not expressed. Of the 117clones screened, 8 clones had a knockout of the rearranged allele and 10had a knockout of the germline allele, for an overall frequency of about15% targeting, an expected frequency for DT40. FIG. 6 shows an exampleof results of a screen.

The left panel of FIG. 6 shows results obtained from a knockout. Oneprimer is in the genomic flanking region 5′ of the targeting vector(actgtgctgcaggtggctatg; SEQ ID NO: 53), and the other primer is in theselectable marker cassette (atacgatgttccagattacgctt; SEQ ID NO:54). Thesecond panel of FIG. 6 shows allele-specific PCR. Both primers are inthe chicken light chain locus (forward primer GCGCTGACTCAGCCGTCCTC (SEQID NO:55); reverse primer gagacgaggtcagcgactcac (SEQ ID NO:56)) andproduce a smaller product from the rearranged allele (R allele) becausethe VJ intron has been deleted from that allele; the germline allele (Gallele) contains the intron and produces a larger fragment. In theknockout of the germline allele (KO-G) only the R allele is detected. Inthe knockout of the rearranged allele (KO-R) only the G allele isdetected. Third set of panels in FIG. 6 shows results obtained for theknock-in. The 5′ assay detects the β-actin-neo fusion on the 5′ side ofintegration (forward primer ctctgctaaccatgttcatgccttc (SEQ ID NO:57);reverse primer AGTGACAACGTCGAGCACAGCT (SEQ ID NO:58)). The 3′ assayemploys two primers in the light chain spanning the attR site (forwardprimer cgcacacgtataacatccatgaa (SEQ ID NO:59); reverse primergtgtgagatgcagacagcacgc (SEQ ID NO:60)). In knock-in samples, both thewild type allele and knock-in allele are detected, whereas in wild typesamples only the wild type fragment is observed. The fourth panel ofFIG. 6 shows RT-PCR results that show expression of the huVK-chCLchimeric light chain in two knock-in DT40 clones (KI) (huVK reaction:forward primer ATGGAAGCCCCAGCTCAGCTTC (SEQ ID NO:61); reverse primercaggtagctgctggccatatac (SEQ ID NO:62); B-actin reaction forward primeraacaccccagccatgtatgta (SEQ ID NO:63); B-actin reaction reverse primertttcattgtgctaggtgcca (SEQ ID NO:64)). Control sample was the parentalknockout (KO).

Example 7 Knock-In Vectors and Transfection of the Same in DT40 Cells

The functional V and pseudogene array was assembled from several chickenand human Ig sequences (FIG. 7). The vector includes a functional,rearranged human V kappa gene (huVK), a chicken light chain constantregion, an array of synthetic VK pseudogenes (SynVK) and chicken intronsand regulatory sequences for proper expression of the light chain. Thefunctional VK fulfills several criteria, both for downstreammanufacturing capability and in order to support B cell development inthe chicken. The functional VK and VH used should express at highlevels, fold into the proper structure, pair with each other efficientlyto form a functional antibody molecule, and not recognize any chickenepitopes which would lead to self-reactive B cells in the chicken.

To select a functional pair of human VK and VH genes for insertion, anumber of rearranged, functional human Vs were cloned from human B cellDNA. 16 VK and 16 VH genes were then expressed in combinations to find apair that would form a functional antibody that expresses at highlevels. The selected VK sequence, clone E6, was identical to thegermline gene V_(K)3-15 except for 3 amino acid changes in frameworkregion 1. The SynVK pseudogene array was designed to have frameworkregions identical to huVK E6, and CDRs that contain tyrosine andtryptophan. The SynVK genes were synthesized and assembled into an arrayof 12 pseudogenes. The functional human VK was synthesized with chickenintron sequences and was then cloned with the chicken light chainpromoter, constant region and J-C intron. The resulting knock-in locuswill express a chimeric light chain consisting of fully human V regionspliced to the chicken light chain constant region, using chickennon-coding, regulatory sequences. Finally, for insertion of the vectorinto the knockout allele we added an attB site and β-actin promoter, anda loxP site was included for eventual excision of the selectable markersand plasmid backbone. The knock-in strategy is illustrated in FIG. 7.

The SynVK insertion vector was designed to enable a simple surface IgM(sIgM) reversion assay for gene conversion (Buerstedde, Reynaud et al.1990. Light chain gene conversion continues at high rate in anALV-induced cell line Embo J 1990 vol. 9 (3) pp. 921-7). A stop codonwas introduced into the CDR1 of the “functional” expressed human Vregion, so that full-length light chain will not be expressed. With nolight chain present, the DT40 heavy chain will not traffic to the cellsurface and the knock-in cells will be sIgM negative. Gene conversion inCDR1 by the SynVK pseudogenes, which do not contain the stop codon, willrepair the light chain sequence and restore its full-length open readingframe. The light chain can then bind to the heavy chain to form the fullIgM complex, and the cells will become sIgM-positive. The DT40 knock-inclones can be stained for sIgM expression with a mouse anti-chicken IgMantibody (Southern Biotechnology Associates) and sorted for thesIgM-positive cells by flow cytometry to obtain a pure population ofgene converted cells for detailed analysis of the gene conversion at thesequence level. A version of the vector with a fully wild type E6 VKregion was also made to verify that the chimeric light chain (human Vregion+chicken C region) is expressed well and can pair with the DT40heavy chain; goat anti-human kappa antibodies verified the expression ofthe human variable region on the surface of transfected DT40.

The SynVK insertion vector was co-transfected with a CMV-integraseexpression construct into IgL knockout DT40 cells. Both constructs wereintroduced as circular, supercoiled DNAs. The knockout cells wereexpected to be sensitive to G418 selection (neomycin) because the neogene in the knockout allele lacks a promoter, and only after insertionof the SynVK vector, linking the β-actin promoter to the neo gene, wouldG418 resistance be induced. After transfection of the SynVK vector,cells were selected for insertion in 4 or 6 mg/ml G418 (a relativelyhigh level of drug selection was required because of some backgroundneo-resistance observed in the knockout cells). Colonies were obtained,and two clones out of eight were found to contain the knock-in, usingPCR assays for the 5′ and 3′ sides of the insertion and for the human VKfunctional gene. FIG. 2 shows PCR results for one of the clones.

Example 8 Demonstration of Gene Conversion of the Synthetic V Regions inDT40 Cells

Light chain knock-in DT40 clones were propagated for several weeks aftertransfection to allow time for the accumulation of gene conversionevents leading to repair of the stop codon and concurrent insertion oftyrosine and tryptophan in CDR1. Cells were sampled at severaltimepoints and stained for sIgM expression using the mouse anti-chickenIgM antibody. In knock-in clones about two weeks post-transfection, fewif any cells expressed sIgM. At 29-41 days post-transfection, a smallpopulation (0.2%) of sIgM-positive cells was observed which allowed usto sort cells by FACS for PCR and sequence analysis. Cell lines with anearly version of the knock-in construct containing only two pseudogenes(2-SynVK) were analyzed, as well as the version with 12 pseudogenes(12-SynVK).

Genomic DNA was prepared from sorted sIgM+ cells and the functional huVKgene was amplified by PCR using primers in the human VK leader sequenceand in the intron downstream of the huVK. PCR products were cloned andsingle colonies were picked for minipreps and sequencing. Good qualitysequence was obtained from about 350 PCR clones. Clear gene conversionthat could be unequivocally assigned to a pseudogene donor was observedin 86 clones with 2-SynVK, and 157 clones for 12-SynVK (FIG. 8). Eachgene conversion event created a synthetic CDR1 in the functional Vcontaining either tyrosine or tryptophan. Since repair of the stop codonin CDR1 was selected, it follows that many of the gene conversion wasobserved only in CDR1. In 4 sequences, gene conversion was also observedin CDR2, likely the result of a single, long gene conversion eventbecause the same pseudogene that converted CDR1 also converted CDR2 ineach case. Several sequences contained clear gene conversion, but it wasimpossible to assign a specific pseudogene donor; these sequences couldbe the result of two overlapping gene conversion events, or a shortaborted gene conversion by a single pseudogene. No evidence for geneconversion by a chicken VL pseudogene was observed; all of the geneconversion tracts were derived from the SynVK pseudogene pool. Somepoint mutations were also observed, which may have been introduced byDT40 or by Taq polymerase errors. However, one sequence had repaired thestop codon by a point mutation, which is likely a DT40-derived mutationbecause it was selected for sIgM expression.

In both 2-SynVK and 12-SynVK containing cells, all of the SynVpseudogenes participated in gene conversion, except for pseudogeneSynVK9 (FIG. 8). With 2-SynVK, the proximal pseudogene was utilizedabout 9 times more than the distal pseudogene. The two pseudogenes arealso in different orientations relative to the functional VK, theproximal pseudogene being in reverse orientation, which raised thequestion of whether proximity or orientation were more important indetermining the efficiency of a pseudogene to participate in geneconversion. In the 12-SynVK cells, proximity to the functional VK didnot seem to influence the frequency of gene conversion, as distalpseudogenes and proximal pseudogenes were used at similar frequencies.Thus orientation may be more important than proximity, with a reverseorientation being more efficient.

The 2-SynVK and 12-SynVK containing constructs are diagrammed in FIG.8A, showing the numbered SynVK pseudogenes, the functional huVK gene,and the chicken constant region. The pseudogenes were numbered prior tocloning in the array, arbitrarily, and they were not assembled innumerical order. The orientation of the SynVK and huVK are indicatedwith arrows above or below. The number of times each pseudogene was usedin gene conversion is indicated. The total number of gene conversionevents is slightly higher because some of the observed gene conversionevents could not be assigned to a specific pseudogene. FIG. 8B showsexamples of gene conversion events. The CDR1 sequence of the“functional” huVK is shown (Input). The stop codon underlined. Sequencesobtained from surface IgM+DT40 are shown below, with the SynVKpseudogene that was used in gene conversion indicated to the right.Tyrosine and tryptophan accumulated in all positions of CDR1. Dashesindicate sequence identity with the input sequence.

Pseudogene SynVK9 had no gene conversion and SynVK3 had only one event.These two pseudogenes insert a bulky residue (tyrosine or tryptophan) atthe third codon in CDR1, whereas all of the other pseudogenes insert thewild type valine residue in that position. In this artificial DT40system with its specific heavy chain, it is possible that a light chainwith a bulky residue in that position inactivates that antibody, makingit impossible to recover sIgM-positive revertants with gene conversionby these pseudogenes.

These results discussed above demonstrate gene conversion of a human VKregion in chicken B cells, creating synthetic CDRs containing tyrosineand tryptophan.

Example 9 Identification of Optimal Human VH and VL Frameworks

cDNA was prepared from normal human peripheral blood lymphocytes and PCRamplified with VH3 and V_(K)3 specific primer pairs. The V_(H)3 primerswere 5′ GGCTGCGATCGCCATGGAGTTTGGGCTKAGCTGG 3′ forward (SEQ ID NO: 49) 5′ATGCGTTTAAACTTTACCCGGAGACAGGGAGAGG 3′ reverse (SEQ ID NO: 50). Thisprimer pair amplified a 1.5 kb DNA fragment corresponding to full heavychain of the V_(H)3/IgG1 isotype.

The V_(K)3 primers were: 5′ GGCTGCGATCGCCATGGAACCATGGAAGCCCCAGCAC 3′forward (SEQ ID NO: 51) and 5′ GGGGGTTTAAACACACTCTCCCCTGTTGAAGCTCT 3′reverse (SEQ ID NO: 52). This primer pair amplified a 700 bp DNAfragment corresponding to the full light chain of the V_(K)3/C_(K)isotype.

Amplicons were cloned directly into the expression vector pF4a (Promega)and verified to have functional coding sequence. Thirty unique sequenceheavy chains and thirty unique sequence light chains were used forevaluation of expression levels. In all experiments plasmid DNA wascarefully quantified and used in transient transfection to produce fullhuman IgG protein, which was then quantified by ELISA. First, a singlefunctional heavy chain was paired with each of the 30 light chains. Inparallel a single light chain was paired with each of the 30 functionalheavy chains. This allowed the selection of the top 16 expressing heavychains and top 16 light chains to generate four 8×8 matrices. An examplematrix is shown in FIG. 9.

The top 2 heavy and light chains were selected from these matrices andwere further analyzed in transient transfection by varying the plasmidDNA concentrations. Also, the relative stability of the different pairswas evaluated by extended incubation at 37° C. prior to proteinquantification. The results of these experiments shown in FIG. 10clearly demonstrate that the optimal pair for expression level andstability is clone “E6” light chain and clone “C3” heavy chain. Theframework regions of these V genes were therefore used as the basis forthe construction of the SynV light chain and heavy chain loci,respectively. The nucleotide sequences of the E6 and C3 clones and theencoded amino acid sequences are shown in FIGS. 11 and 12.

What is claimed is:
 1. A transgenic animal that develops its primaryantibody repertoire by gene conversion, wherein the transgenic animalcomprises a genome comprising a recombinant immunoglobulin heavy chain(IgH) locus comprising: a) a functional (IgH) gene comprising a nucleicacid encoding a heavy chain variable region comprising: i) heavy chainCDR1, CDR2 and CDR3 regions; and ii) a heavy chain framework; and, b) aplurality of pseudogenes that encode heavy chain variable regions eachcomprising: i) heavy chain CDR1, CDR2 and CDR3 regions; and ii) a heavychain framework region that is identical in amino acid sequence to theheavy chain framework of a) (ii); wherein said recombinant IgH locuscomprises: in operable linkage: an intron region, a constantregion-encoding region and a 3′ untranslated region; wherein at leastpart of said intron region is endogenous to the genome of saidtransgenic animal; and said nucleic acid of a) and pseudogene of b), areexogenous to the genome of said transgenic animal, wherein each aminoacid residue of the CDRs encoded by the pseudogenes of b) and each aminoacid residue of the CDRs of the heavy chain variable region of a) areselected from the same group of 2 to 5 amino acid residues, wherein saidplurality of pseudogenes are operably linked to said functional IgH geneand donate nucleotide sequences to the nucleic acid of a) by geneconversion in said transgenic animal; and wherein said transgenic animalexpresses a recombinant immunoglobulin comprising a diversified form ofsaid functional IgH variable region.
 2. The transgenic animal of claim1, wherein: at least one of the 2 to 5 amino acids in said group is atyrosine or tryptophan residue, and at least one of the 2 to 5 aminoacids in said group is an alanine, glycine or serine residue.
 3. Thetransgenic animal of claim 1, wherein said heavy chain framework isidentical to a human framework.
 4. The transgenic animal of claim 1,wherein said heavy chain framework is least 95% identical to a humangermline framework.
 5. The transgenic animal of claim 1, wherein saidheavy chain framework is from the animal.
 6. The transgenic animal ofclaim 1, wherein said heavy chain framework is a humanized framework. 7.The transgenic animal of claim 1, wherein the constant region is a humanconstant region.
 8. The transgenic animal of claim 1, wherein saidimmunoglobulin heavy chain locus comprises: in operable linkage: anintron region, a constant region-encoding region and a 3′ untranslatedregion; wherein said intron region, said constant region-encoding regionand said 3′ untranslated region are endogenous to the genome of saidtransgenic chicken; and said nucleic acid of a) and pseudogenes of b),wherein said nucleic acid of a) and pseudogenes of b) are exogenous tothe genome of said transgenic chicken.
 9. The transgenic animal of claim1, wherein said immunoglobulin heavy chain locus comprises at least 10of said pseudogenes.
 10. The transgenic animal of claim 1, wherein atleast one of said plurality of pseudogenes of b) is in animalorientation relative to the nucleic acid of a).
 11. The transgenicanimal of claim 1, wherein the transgenic animal is a chicken.
 12. Amethod comprising: (A) immunizing a transgenic animal of claim 1 with anantigen, and (B) obtaining from said transgenic animal an antibody thatspecifically binds to said antigen, or a B cell that produces the same.13. The method of claim 12, further comprising: making hybridomas usingcells of said transgenic animal; and screening said hybridomas toidentify a hybridoma that produces an antibody that specifically bindsto said antigen.
 14. The method of claim 12, further comprisingamplifying the heavy and light chain variable region-encoding nucleicacid from lymphocytes of said transgenic animal, introducing theamplified nucleic acid into a cell and expressing the nucleic acid insaid cell, thereby producing a recombinant antibody comprising the heavyand light chain variable regions of the antibody.
 15. The method ofclaim 12, wherein the antibody is humanized.
 16. The method of claim 12,wherein the animal is a chicken.
 17. An isolated B cell of thetransgenic animal of claim 1, wherein the B cell secretes an antibody.18. The isolated B cell of claim 17, wherein the antibody ispost-translationally modified by the B cell.
 19. The isolated B cell ofclaim 18, wherein the antibody is glycosylated by the B cell.
 20. Theisolated B cell of claim 17, wherein the isolated B cell is a chicken Bcell.